Use of cyclodextrin for treatment and prevention of bronchial inflammatory diseases

ABSTRACT

The invention provides the use of a cyclodextrin compound for the manufacturing of a medicament for the treatment or prevention of bronchial inflammatory diseases, particularly for asthma.

The present invention relates to the use of cyclodextrin compound forthe treatment and prevention of bronchial inflammatory diseases.

Compounds for the treatment and prevention of bronchial inflammatorydiseases are classified in the art as bronchodilator also calledreliever medications or nonbronchodilators antiinflammatory agentsreferred to as controller agents, on the basis of their pharmacodynamiceffects. Short-acting bronchodilators such as inhaled beta agonist oranticholinergics are considered reliever medications. Corticosteroids,cromolyn sodium, nedocromil sodium, sustained-release theophylline andlong-acting beta agonist are considered controller medications, sincethey are used to achieve and maintain control of symptoms and are useddaily on a long-term basis.

Among reliever medication, inhaled β2-adrenergic agonists are drugs forrelief of symptoms due to acute airway obstruction. They have a rapidonset of action and a 3-6 h duration of activity. Unfortunately theyhave side effects such as tachycardia, palpitations and tremor thatoften disappear during chronic administration.

Anticholinergic agents induce airway smooth muscle relaxation. Theiractivity is not as effective as beta agonists in asthma but is moreprolonged (6 to 8 hours).

Among controller medications, glucocorticosteroids are effective agentswith anti-inflammatory effects. Unfortunately, their side effectsinclude adrenal suppression, osteoporosis, growth suppression, weightgain, hypertension, diabetes, dermal thinning, cataracts, myopathy andpsychotic actions. These effects are dose related and are usually seenwith systemic administration. Local side effects, including oralcandidiasis and dysphonia may occur at lower doses of inhaledglucocorticoids.

Cromolyn sodium and nedocromil sodium are also classified as controlleragents, because of their similar clinical profile. They inhibitbronchoconstriction induced by neurally mediated events.

Theophylline is generally considered as a bronchodilator but has weakbronchodilator activity in therapeutic doses. It may also haveanti-inflammatory properties. The dose-related adverse effects oftheophylline are nausea, nervousness, anxiety and tachycardia.

Lipoxygenase inhibitors and leukotriene receptor agonists are alsocontroller agents. They alter the pathological effects of leukotrienesderived from the 5-lipoxygenation of arachidonic acid. They can inhibitthe bronchospastic effects of allergens, exercise, cold dry air, andaspirin allergy. Both are efficaceous in alleviating symptoms andimproving pulmonary function during 4-6 weeks of therapy in patientswith moderate asthma.

There is therefore a need for improved compounds which can be used forthe treatment or prevention of bronchial inflammatory diseases.

It is now surprisingly found that cyclodextrin is useful as activecomponent for the treatment or prevention of bronchial inflammatorydiseases.

The invention therefore provides the use of cyclodextrin compound forthe treatment or prevention of bronchial inflammatory disease in a hostmammal in need of such treatment.

By cyclodextrin compound, one means cyclodextrin as well as theirpharmaceutically acceptable salts, enantiomeric forms, diastereoisomersand racemates.

By cyclodextrin, one means cyclic oligosaccharides produced by enzymaticdegradation of starch such as described in “Cyclodextrin Technology, JSzejtli, Kluwer Academic Publishers 1998, pp 1-78”, and which arecomposed of a variable number of glucopyrannose units (n), mostly 6, 7or 8. These cyclodextrins are respectively named α, β and γcyclodextrins (αCD, βCD, γCD).

Cyclodextrin is also represented by CD hereafter.

Cyclodextrin compound according to the invention is cyclodextrin per se,alkyl-cyclodextrin (R-CD) wherein R is methyl, ethyl, propyl and butyl;carboxyalkyl-cyclodextrin (CR-CD), etherified-cyclodextrin (RO-CD),sulfoalkyl-cyclodextrin (SR-CD), hydroxyalkyl-cyclodextrin (HR-CD),glucosyl-cyclodextrin, di and triglycerides-cyclodextrin or acombination thereof and their pharmaceutically acceptable salts whichare at least water soluble in an amount of 0.5 gr/100 ml at 25° C.

The water-soluble cyclodextrin compound preferably used in the presentinvention refers to a cyclodextrin compound having water solubility ofat least that of β-cyclodextrin (1.85 g/100 ml). Examples of suchwater-soluble cyclodextrin compound are sulfobutylcyclodextrin,hydroxypropylcyclodextrin, maltosylcyclodextrin, and salts thereof. Inparticular, sulfobutyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin,maltosyl-β-cyclodextrin, and salts thereof

Other preferred cyclodextrin compound according to the invention aremethylcyclodextrins (products of the cyclodextrins methylation) such as2-O-methylβ-cyclodextrin;

-   -   dimethylcyclodextrin (DIMEB) (preferably substituted in 2 and in        6); trimethylcyclodextrin (preferably substituted in 2, 3 and        6);    -   “random methylated” cyclodextrins (RAMEB or RM) (preferably        substituted at random in 2, 3 and 6, but with a number of 1,7 to        1,9 methyl by unit glucopyrannose), hydroxypropylcyclodextrins        (HPCD), hydroxypropylated cyclodextrins preferably substituted        randomly mainly in position 2 and 3 (HP-βCD, BP-γCD)),        sulfobutylethercyclodextrins (SBECD),        hydroxyethyl-cyclodextrins, carboxymethylethylcyclodextrins,        ethylcyclodextrins, cyclodextrins amphiphiles obtained by        grafting hydrocarbonated chains in the hydroxyl groups and being        able to form nanoparticles, cholesterol cyclodextrins and        triglycerides-cyclodextrins obtained by grafting cyclodextrins        monoaminated (with a spacer arm) as described in Critical Review        in Therapeutic drug Carrier Systems, Stephen D. Bruck Ed,        Cyclodextrin-Enabling Excipient; their present and future use in        Pharmaceuticals, D. Thomson, Volume 14, Issue 1 p 1-114 (1997)

Most preferred cyclodextrins compounds are

-   -   β-cyclodextrin with optionally a chemical function grafted on        the glucopyrannose units such as hydroxypropyl-βcyclodextrin        (HPPCD), sulfonylbutylether-βcyclodextrin (SBEβCD), random        methylated-βcyclodextrin (RMβCD), dimethyl-βcyclodextrin        (DIMEβCD), trimethyl-βcyclodextrin (TRIMEβCD), hydroxybutyl        βcyclodextrin (HBβCD), glucosyl βcyclodextrin, maltosyl        βcyclodextrin and 2-O-methyl βcyclodextrin (Crysmeb), or a        combination thereof and their pharmaceutically acceptable salts.

The cyclodextrin compounds according to the invention are produced bythe well-known enzymatic degradation of starch such as the methoddescribed in “Cyclodextrin Technology, J Szejtli, Kluwer AcademicPublishers 1998, pp 1-78, followed by grafting of an appropriatechemical group.

The invention further provides the use of such cyclodextrin compound forthe manufacturing of a medicament for the treatment or prevention ofbronchial inflammatory diseases to a patient in need of such treatment.

According to the invention the cyclodextrin compound has to beadministered to the patient over several months or years (especially incase of prevention). The cyclodextrin compound is administeredpreferably as aerosol, with non-toxic doses ranging between nanomolarand molar concentrations.

The invention relates to a method used for treating bronchialinflammatory diseases, preferably asthma and chronic obstructivepulmonary disease (COPD) in a host mammal in need of such treatment,e.g., a patient suffering from such a disease, by the application of acyclodextrin compound according to the invention in a pharmaceuticallyeffective amount. Asthma is an inflammatory disease of the bronchialtree related or not to an allergen exposure. This inflammation provokessymptoms in patients by stimulating the bronchial smooth muscles tocontract, enhancing the mucus secretion, and inducing bronchialmorphological changes thought to be an aggravating factor regarding thecourse of the disease. Airway hyperresponsiveness is a hallmark of thedisease and is responsible for most of symptoms. Bronchial tree is avery complex tissue with many cell types (as for example epithelialcells, smooth muscle cells, inflammatory cells, nerves, mucus producingcells, fibroblasts) and the bronchial remodelling events which comprisemany aspects mainly consist in a deposition of extracellular matrixcomponents in the bronchial walls, a smooth muscle hyperplasia and ahyperplasia of the mucus producing cells. The use of cyclodextrincompounds according to the invention inhibits the inflammatory cellsinflux in the compartments of bronchoalveolar lavage and peribronchialtissue and inhibits the hyperresponsiveness defined as an abnormalresponse to stimulating agents such as methacholine. The disease andcurrent treatments are reviewed in, e.g., GINA Workshop Report, GlobalStrategy for Asthma Management and Prevention (NIH Publication No.02-3659) and Fabbri, L. M., and Hurd, S. S., Eur. Respir. J. 22 (2003)1-2.

The invention therefore further relates to a method for treatingbronchial inflammatory diseases in a patient suffering from such adisease, using a cyclodextrin compound according to the invention in atherapeutically effective amount.

The invention preferably further relates to a method for treatingemphysema in a patient suffering from such a disease, using cyclodextrincompounds according to the invention. In such a disease, the alveolarwalls are destroyed by proteolytic processes and this destructionimpairs the transfer of oxygen to the blood. In such a disease,physiological problems also occurs because of the derived hyperinflationwhich causes abnormalities in the ventilation by causing a dysfunctionof respiratory muscles and because of a hypertension in pulmonaryarteries leading to cardiac failure in advanced stages.

The invention preferably further relates to a method for treatingchronic obstructive pulmonary disease (COPD) in a patient suffering fromsuch a disease, using cyclodextrin compounds according to the invention.In such a disease, the bronchial walls of small airways are remodelledby proteolytic processes and this remodelling and fibrosis induce anairway obstruction which can be measured by spirometry. In such adisease, physiological problems also occurs because of the derivedhyperinflation which causes abnormalities in the ventilation/perfusionratio and causes hypoventilation and eventually CO2 accumulation.

According to the invention the cyclodextrin compound has to beadministered over several months or years, to the patient in need ofsuch a therapy. The cyclodextrin compounds are administered preferablyby the aerosolization of a liquid or powder composition, with non-toxicdoses ranging between micro and molar concentrations per kg and day.

A further preferred object of the invention is a pharmaceuticalcomposition of cyclodextrin compound according to the invention for thetreatment of bronchial inflammatory diseases, and its use, containing acyclodextrin or a salt thereof and preferably a water-solublecyclodextrin derivative (water soluble being defined as a solubility ofat least 0.5 g/100 ml water at 25° C.).

The pharmaceutical compositions are aqueous compositions havingphysiological compatibility. The compositions include, in addition tocyclodextrin or a salt thereof, auxiliary substances, buffers,preservatives, solvents and/or viscosity modulating agents. Appropriatebuffer systems are based on sodium phosphate, sodium acetate or sodiumborate. Preservatives are required to prevent microbial contamination ofthe pharmaceutical composition during use. Suitable preservatives are,for example, benzalkonium chloride, chlorobutanol, methylparabene,propylparabene, phenylethyl alcohol, sorbic acid. Such preservatives areused typically in an amount of 0.01 to 1% weight/volume.

The cyclodextrin compound of the present invention exhibits its effectsthrough either oral administration, parenteral administration or topicaladministration, and it is preferably formed into a composition forparenteral administration, particularly an injection composition ortopical administration, particularly an aerosol composition. Suchaerosol composition is for example a solution, a suspension, amicronised powder mixture and the like. The composition is administeredby using a nebulizer, a metered dose inhaler or a dry powder inhaler orany device designed for such an administration.

Examples of galenic compositions include tablets, capsules, powders,granules and the like. These may be produced through well knowntechnique and with use of typical additives such as excipients,lubricants, and binders.

Suitable auxiliary substances and pharmaceutical compositions aredescribed in Remington's Pharmaceutical Sciences, 16th ed., 1980, MackPublishing Co., edited by Oslo et al. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the composition torender the composition isotonic. Examples of pharmaceutically acceptablesubstances include saline, Ringer's solution and dextrose solution. pHof the solution is preferably from about 5 to about 8, and morepreferably from about 7 to about 7.5.

A preferred pharmaceutical composition for nebulization comprisescyclodextrin (CD), NaCl and water.

The solution is prepared by dissolving CD in 100 ml of purified water,adding NaCl by stirring so as to dissolve them and complete with waterso as to obtain 200 ml of solution. Preferably the solution issterilized by filtration through a 0.22 μm polypropylene membrane or bya steam sterilization process.

Especially preferred composition is a combination of (for 200 ml ofsolution): 10-50 g CD, preferably 20 gCD, preferably HPβCD; sodiumchloride 1.2-1.5 g, preferably 1.42 g (isotonicity) and water,preferably pyrogen-free, sterile, purified water ad 200 ml.

Such a composition is useful for the treatment of bronchial inflammatorydiseases.

Most preferred composition is a combination of 2-O-methylβCD with sodiumchloride 1.2-1.5 g, preferably 1.42 g (isotonicity) and water,preferably pyrogen-free, sterile, purified water ad 200 ml.

The following examples, references, and figures are provided to aid theunderstanding of the present invention. It is understood thatmodifications can be made in the procedures set forth without departingfrom the invention.

DESCRIPTION OF THE FIGURES

FIG. 1-2 Effects of inhalation of cyclodextrin compound on BALeosinophil percentage (FIG. 1) and peribronchial inflammation score(FIG. 2). Controls are mice exposed to ova by inhalation and placebo byinhalation (OVA)

FIG. 3 Effects of inhalation of cyclodextrin compound on peribronchialeosinophils reported here as a number/mm of epithelial basementmembrane.

FIG. 4 Airway responsiveness measurements: Enhanced Pause (Penh) wasmeasured in OVA exposed mice during 5 minutes after a 2 minutesinhalation of cyclodextrin or Placebo (OVA) and increasing doses ofmethacholine (Mch).

FIG. 5 Measurement of cytokines by Elisa. Eotaxin was measured byincubation in a wheel coated with a primary antibody specificallydedicated to the recognition of the protein and after rinsing, a secondantibody against eotaxin, coupled with horse radish peroxydase was usedto quantify the amounts of eotaxin in the solution.

FIG. 6 Measurement of allergen specific IgE levels in serum.

FIG. 7 Measurement of eotaxin and IL-13 in Bal and lung proteinextracts. IL-13 was measured by incubation in a wheel coated with aprimary antibody specifically dedicated to the recognition of theprotein and after rinsing, a second antibody against IL-13, coupled withhorse radish peroxydase was used to quantify the amounts of eotaxin inthe solution.

FIG. 8 Airway responsiveness measurements: Enhanced Ponse (Penh) wasmeasured in mouses after receiving crysmeb or placebo treatment

FIG. 9 Peribronchial inflammation score measured in histology whentreated with various Crysmeb concentration compared to placebo (FIG. 9A)and to other cyclodextrin and fluticasone FIG. 9B)

FIG. 10 Airway responsiveness measurements: comparison of cyclodextrincompounds with placebo and Fluticasone. Measurements ofmethacholine-induced airway response in mice exposed 7 days to allergensand receiving an inhaled therapy 30 min before the allergen exposition.

FIG. 11 Levels of IL-13 measured by Elisa in lung protein extracts.

EXAMPLE 1 Use of Compositions Containing HP-β-Cyclodextrin for Therapyof Allergen-Induced Airway Inflammation and BronchialHyperresponsiveness in a Mouse Model of Asthma Materials

BP-β-CD (degree of substitution=0.64) was obtained from Roquette(France). α- and HP-γ-D were obtained from Wacker Chemie Gmbh (Germany).Apyrogenic phosphate buffered saline (PBS) was purchased fromBio-Wittaker (Verviers, Belgium). Methacholine was from Sigma-Aldrich(Germany).

All other materials were of analytical grade. Sterile water forinjection was used throughout this study. Sterile, apyrogenic andisotonic CD solutions were prepared at 1, 7.5 and 50 mM for BP-β-CD andα-CD and at 50 mM for HP-γ-CD. Cyclodextrins were tested following theBacterial Endotoxin Test described in USP XXVI using Limulus AmebocyteLysate (LAL). Osmolalities of all the solutions were measured by aKnauer Automatic semi-micro Osmometer and adjusted to the value of 300mOsm/kg by the addition of an adequate amount of NaCl. A terminalsterilization of the solutions was performed by steam sterilizationprocess.

Methods

Aerosol was produced by using an ultrasonic nebuliser SYSTAM, thevibration frequency of which is 2.4MHz with variable vibration intensityand ventilation levels. Vibration intensity was fixed in position 6 andthe ventilation level was 25 (t½) l/min.

Characterization of Nebulized Aerosol

Aerosol size distribution emitted from CDs solutions was determined witha laser size analyzer Mastersizer (Malvern, Orsay, France). Tenmilliliters of each solution were directly nebulized in the laser beam.The mouth piece was held at 1 cm from the center of the laser beam. Theresulting aerosol was aspirated on the opposite side of the beam.Environmental temperature and relative humidity were maintainedconstant, that is to say at 20° C. and 40-45%. Triplicates of eachmeasurement were performed and compared to controls of PBS. The resultsare expressed as the percentage of droplets comprised in the range 0.5to 5.79 μm and the median diameter. The concentration of droplets in theair evaluated by the obscuration percentage of the laser beam was in thesame range for each experiment (15-25%). A comparison of the MMAD, theGSD and the percentage of droplets comprised in the range of 0.5 to 5.79μm of all the CDs solutions with the corresponding values for PBSdemonstrated that the presence of CDs in the solution did not influencethe droplet size distribution in the aerosols. A fraction of dropletscomprised in the range of 0.5 to 5.79 μm close to 65% was obtained ineach experiment.

Sensitisation, Allergen Exposure and Therapeutic Protocols.

In order to study modulation of airway inflammation males BALB/c mice of6 to 8 weeks old were sensitized by intraperitoneal injection of 10 μgovalbumin (OVA) (Sigma Aldrich, Schnelldorf, Germany) emulsified inaluminum hydroxyde (AlumInject; Perbio, Erembodegem, Belgium) on days 1and 8. Mice were subsequently exposed to allergens by daily inhalationof an aerosol of OVA 1%, for 30 minutes, generated by ultrasonicnebulizer (Devilbiss 2000), from day 21 to 27. Mice were subjected toinhalation of α-CD, HP-β-CD 1, 7.5, 50 mM and HP-γ-CD 50 mM 30 minutesbefore OVA inhalation. Mice were sacrificed performed on day 28 aspreviously reported by Cataldo and al in Am. J. Pathol 2002;161(2):491-498.

Bronchoalveolar Lavage Fluid (BAL)

Immediately after assessment of airway responsiveness, and 24 hoursafter the last allergen exposure.

Mice were sacrificed and a bronchoalveolar ravage was performed using4×1 ml PBS-EDTA 0.05 mM (Calbiochem, Darmstadt, Germany) as previouslydescribed by Cataldo D D, Tournoy K G, Vermaelen K et al. in Am J Pathol2002; 161(2):491-498. Cells were recovered by gentle manual aspiration.After centrifugation of bronchoalveolar fluid (BALF) (1200 rpm for 10minutes, at 4° C.), the supernatant was frozen at −80° C. for proteinassessment and the cell pellet was resuspended in 1 ml PBS-EDTA 0.05 mM.The differential cell counts were performed on cytocentrifugedpreparations (Cytospin) after staining with Diff-Quick (Dade, Belgium).

Pulmonary Histology and Tissue Processing

After BAL, the thorax was opened and the left main bronchus was clamped.The left lung was excised and frozen immediately at −80° C. for proteinand mRNA extraction. The right lung was infused with 4 mlparaformaldehyde 4%, embedded in paraffin and used for histology.Sections of 5 μm thickness were cut off from paraffin and were stainedwith haematoxylin-eosin. The extent of peribronchial inflammation wasestimated by a score calculated by quantification of peribronchialinflammatory cells, as previously described by Cataldo D D, Tournoy K G,Vermaelen K et al. in Am J Pathol 2002; 161(2):491-498. A value of 0 wasadjudged when no inflammation was detectable, a value of 1 when therewas occasionally inflammatory cells, a value of 2 when most bronchi weresurrounded by a thin layer (1 to 5 cells) of inflammatory cells and avalue of 3 when most bronchi were surrounded by a thick layer (>5 cells)of inflammatory cells. Since 5-7 randomly selected tissue sections permouse were scored, inflammation scores are expressed as a mean value andcan be compared between groups. After Congo Red staining, theeosinophilic infiltration in the airway walls was quantified by manualcount and reported to the perimeter of epithelial basement membranedefining an eosinophilic inflammatory score.

The left lung was crushed using a Mikro-Dismembrator (Braun BiotechInternational, Gmbh Melsungen, Germany). For proteins extraction, thecrushed lung tissue was incubated overnight at 4° C. in a solutioncontaining 2M urea, 1M NaCl and 50 mM Tris (pH 7.5) and subsequentlycentrifuged 15 minutes at 16.000×g. The supernatant was stored at −80°C.

Bronchial Responsiveness Measurement

Twenty-four hours after the last allergen exposure, the bronchial hyperresponsiveness was determined by measuring the Penh (Enhanced Pause)using a barometric plethysmograph (Emka technologies, Paris) as proposedby Hamelmann, E., et al., Am. J. Respir. Crit. Care Med. 156 (1997)766-775). The Penh was measured at baseline and 5 min after theinhalation of increasing doses (25, 50, 75 and 100 mM) of methacholine(Mch).

Measurements of Cytokines by ELISA

Eotaxin and IL-13 levels were assessed using commercial ELISAs (R&Dsystems, Abingdon, UK).

Eotaxin was measured by incubation in a wheel coated with a primaryantibody specifically dedicated to the recognition of the protein andafter rinsing, a second antibody against eotaxin, coupled with horseradish peroxydase was used to quantify the amounts of eotaxin in thesolution

Measurement of Allergen Specific Serum IgE

At the end of the experiment, blood was drawn from the heart formeasurement of OVA specific serum IgE. Microtiter plates were coatedwith OVA. Serum was added followed by a biotinylated polyclonal rabbitanti-mouse IgE (S. Florquin, ULB, Brussels, Belgium). A serum pool fromOVA-sensitized animals was used as internal laboratory standard; 1 unitwas arbitrarily defined as 1/100 dilution of this pool.

Measurement of Eotaxin and IL-13 in Bal and Lung Protein Extracts.

IL-13 was measured by incubation in a wheel coated with a primaryantibody specifically dedicated to the recognition of the protein andafter rinsing, a second antibody against IL-13, coupled with horseradish peroxydase was used to quantify the amounts of eotaxin in thesolution

Statistical Analysis

Results of BAL cell count, pulmonary histology, cytokines and mRNAlevels were expressed as mean+/−SEM and the comparison between thegroups was performed using Mann-Whitney test. Mann-Whitney test wasperformed using GRAPHPAD INSTAT version 3.00 for WINDOWS 95 (GRAPHPADSOFTWARE, San Diego, Calif., USA, WWW.GRAPHPAD.Com.). P values <0.05were considered as significant.

Pharmacological Results: Inflammatory Cells in the BAL.

After allergen exposure, eosinophil counts were significantly decreasedafter the inhalation of HP-β-CD and HP-γ-CD at the dose of 50 mM. Therewas a dose dependent decrease in BAL eosinophils with the HP-β-CD 1, 7.5and 50 mM inhalation. Other inflammatory cells were not present indifferent amounts in the BAL after HP-β-CD inhalation when compared toplacebo. On the contrary, α-CD inhalation led to a tendency to increasethe number of eosinophils in BAL after allergen exposure (FIG. 1).

Peribronchial Inflammation

After allergen exposure, mice treated with placebo displayed asignificant increase in peribronchial inflammation as quantified by theperibronchial inflammation score. Mice treated with HP-β-CD 1, 7.5, and50 mM and HP-γ-CD 50 mM were shown to have decreased inflammation scorewhen compared to placebo treated mice. α-CD inhalation did not reducethe peribronchial inflammation score (FIG. 2).

Peribronchial Eosinophil Infiltration

As demonstrated previously, the allergen exposure did induce asignificant increase in the number of eosinophils detectable in theperibronchial area All CD tested induced a decrease of this infiltrationand this decrease reached statistical significance for α-CD, HP-β-CD 1,7.5, and HP-γ-CD 50 mM (FIG. 3).

Bronchial Responsiveness

The inhalation of HP-β-CD 50 mM reduced the methacholine-induced Penhincrease (FIG. 4). When measuring the area under the methacholinedose-response curve (A.U.C) for different CDs, HP-β-CD 50 mM was theonly to show a significant decrease (FIG. 5).

Cytokine Measurements in BAL and Lung Protein Extracts

When compared to placebo exposed mice, all doses of HP-β-CD testedinduced a decrease in levels of eotaxin measured by ELISA in lungprotein extracts (FIG. 7 a). IL-13 levels were decreased in BAL afterHP-β-CD exposure and, on the contrary, were increased after α-CDexposure (FIG. 7 b).

Measurements of Allergen-Specific IgE in Serum

There were no significant differences between the groups for theallergen sensitization as assessed by the similar levels of OVA specificIgE measured by ELISA in serum (FIG. 6).

EXAMPLE 2 Use of Compositions Comprising 2-O-methyl-cyclodextrin forTherapy of Allergen-Induced Airway Inflammation and BronchialHyperresponsiveness in a Mouse Model of Asthma Materials

Materials are identical to example 1 with the exception of thecyclodextrin compound which is here 2-O-methyl-cyclodextrin, KLEPTOSECRYSMEB®, a product commercialised by Roquette. It has, on average, 4methyl groups per native cyclodextrin molecule and is characterized byan average molecular weight of 1135 and a average molar degree ofsubstitution of 0.57.

Sterile, apyrogenic and isotonic CD solutions were prepared with 10, 20,50 and 75 mM for 2-O-methyl-cyclodextrin. Cyclodextrins were testedfollowing the Bacterial Endotoxin Test described in USP XXVI usingLimulus Amebocyte Lysate (LAL). Osmolalities of all the solutions weremeasured by a Knauer Automatic semi-micro Osmometer and adjusted to thevalue of 280-300 mOsm/kg by the addition of an adequate amount of NaCl.A terminal sterilization of the solutions was performed by steamsterilization process.

Methods

Same methods are used as in example 1 but in the present example we didexpose mice to aerosolized CRYSMEB (10, 20, 50, 100 or 200 mM) in astandard exposure box (20×30×15 cm) for 30 min/day during 7 days.

Pharmacological Results: Inflammatory Cells in the BAL.

The cellular composition of the bronchoalveolar lavage was notsignificantly altered by the exposure to CRYSMEB. In particular, therewere no differences regarding eosinophil and neutrophil counts (seetable 1).

Bronchoalveolar lavage eosinophilia was significantly decreased in thegroups treated by CRYSMEB. The decrease in lavage eosinophilia wascomparable with that obtained with different concentrations ofIP-beta-cyclodextrins or fluticasone, a commonly used inhalation steroidused as a reference therapy (table 2)

Peribronchial Inflammation

After allergen exposure, mice treated with placebo displayed asignificant increase in peribronchial inflammation as quantified by theperibronchial inflammation score. Mice treated with CRYSMEB 20 mM wereshown to have decreased inflammation score when compared to placebotreated mice (FIG. 9A). Peribronchial inflammation score was measuredand was significantly decreased in every treatment group as compared toplacebo (FIG. 9B)

Bronchial Responsiveness

The inhalation of CRYSMEB 10 mM reduced the methacholine-induced Penhincrease (FIG. 10).

The responsiveness to methacholine was increased after allergen exposureand placebo and was significantly reduced by the treatment with CRYSMEBin an extent comparable to that obtained with fluticasone therapy (FIG.10)

Cytokine Measurements in BAL and Lung Protein Extracts

In order to unveil mechanisms implicated in the pharmacological effectof CRYSMEB, we measured IL-13, a major Th2 cytokine implicated in theairway hyperresponsiveness and inflammation. We found that levels ofIL-13 measured by ELISA in whole lung protein extracts weresignificantly decreased by the exposure to CRYSMEB as well asfluticasone and HP-beta-CD 50 mM.

(see FIG. 11) EXAMPLE 3 Pharmaceutical Composition to be Administered inan Aerosol to a Patient in Need of Treatment for Bronchial InflammatoryDisease

HP betaCD 75 mM

Solution osmolality is 308 mOs/kg. pH is 7.2.

The concentration of CD compound can be modified in function of therequirements. It is preferred to adjust the tonicity by addition ofNaCl.

A preferred composition for nebulization is:

For 200 ml of solution:

HPβCD exempt from pyrogenic 20.15 g (75 mM) Sodium chloride  1.42 g(isotonicity) Pyrogen-free, sterile, purified water, q.s. ad 200 ml

-   a) Weigh 20.15 g of HPPCD exempt from pyrogenic (3.2% H₂O, from    ROQUETTE) and dissolve them in 100 ml of purified water.-   b) Weigh 1.42 g of sodium chloride and add them to solution (a) by    energetically stirring so as to dissolve them.-   c) Complete with water so as to obtain 200 ml of solution.

Sterilize by filtration through a 0.22 μm polypropylene membrane.

TABLE 1 differential cell counts in the bronchoalveolar lavage measuredafter the exposure to different concentrations of inhaled CRYSMEB.PLACEBO Crysmeb 20 mM Crysmeb 50 mM Crysmeb 75 mM Epithelial cells (%)15.9714 ± 5.154   29.9 ± 5.909 36.1375 ± 4.52   30.8875 ± 1.349 Eosinophils (%)  0.0428 ± 0.0428 0.0375 ± 0.0375 0.1125 ± 0.0789 0.0375± 0.0375 Neutrophils (%) 0.1285 ± 0.236 0.0375 ± 0.1061   0.2 ± 0.35050.0375 ± 0.1061 Lymphocytes (%)  0.1857 ± 0.1421  0.425 ± 0.1485  0.275± 0.1161  0.075 ± 0.0491 Macrophages (%) 83.5857 ± 1.179*  69.5 ± 5.95663.1625 ± 4.695   68.9 ± 1.334

TABLE 2 differential cell counts in the bronchoalveolar lavage measuredafter the exposure to different concentrations of inhaled CRYSMEB.HPBeta-CD HPBeta-CD HPBeta-CD CRYSMEB PLACEBO Fluticasone 1 mM 10 mM 50mM 10 mM Epithelial  3.86 ± 2.551 22.85 ± 5.343 25.08 ± 3.413 34.44 ±3.723 45.04 ± 5.534 36.83 ± 5.644 cells (%) Eosinophils (%) 53.08 ±4.683  32.92 ± 7.306*  34.2 ± 7.705* 27.24 ± 4.98*  12.18 ± 4.366*  8.84± 2.946* Neutrophils (%)  3.03 ± 1.333  1.85 ± 1.093  0.36 ± 0.1563 0.83 ± 0.5838  0.72 ± 0.3992  1.26 ± 0.4587 Lymphocytes (%)  3.62 ±1.576  1.68 ± 0.7115  0.48 ± 0.1869   0.21 ± 0.08571 0.12 ± 0.12  0.214± 0.1079 Macrophages (%) 36.25 ± 5.016 40.52 ± 3.122 39.75 ± 5.42737.114 ± 3.878  41.86 ± 9.043 52.714 ± 6.49  Total cells 220.42 ± 81.709 75.92 ± 11.922  74.92 ± 14.396 114.43 ± 33.245  37.33 ± 10.683 131.93 ±33.637 (10⁴/ml)

1. A use of a cyclodextrin compound for the manufacturing of amedicament for the treatment of bronchial inflammatory disease in a hostmammal in need of such treatment.
 2. A use of a cyclodextrin compoundfor the manufacturing of a medicament for the prevention of bronchialinflammatory disease in a host mammal in need of such treatment.
 3. Theuse according to claim 1 wherein the cyclodextrin compound has a watersolubility of at least 1.85 g/100 ml.
 4. The use according to claim 1wherein the cyclodextrin compound is a βcyclodextrin compound.
 5. Theuse according to claim 1 wherein the cyclodextrin compound is selectedfrom the group consisting of: β-cyclodextrin,hydroxypropyl-βcyclodextrin, sulfolbutylether-βcyclodextrin, randommethylated-βcyclodextrin, dimethyl-βcyclodextrin,trimethyl-βcyclodextrin, hydroxypropyl βcyclodextrin, hydroxybutylβcyclodextrin, glucosyl-βcyclodextrin, maltosyl-βcyclodextrin,2-O-methyl-βcyclodextrin or a combination thereof and theirpharmaceutically acceptable salts.
 6. The use according to claim 1wherein the cyclodextrin compound is hydroxypropyl β-cyclodextrin
 7. Theuse according to claim 1 wherein the cyclodextrin compound is a2-O-methyl-cyclodextrin.
 8. The use according to claim 1 wherein thebronchial inflammatory disease is asthma.
 9. A method for prevention ofbronchial inflammatory disease comprising the administration to apatient in need of such treatment of an effective dose of cyclodextrincompound.
 10. A method for treatment of bronchial inflammatory diseasecomprising the administration to a patient in need of such treatment ofan effective dose of cyclodextrin compound.